Immunoblot and an enzyme-linked immunosorbent assays were used to evaluate and compare IgG antibodies against S. aureus whole cell lysate, cell wall peptidoglycan and lipoteichoic acid to discriminate between deep-seated and superficial S. aureus infection. Serum samples were examined from patients with deep-seated (n = 25) and superficial (n = 25) S. aureus infections and 15 healthy controls. Patients with deep-seated infections exhibited a large number of immuno-reactive bands in their IgG immunoblot profile as compared to those with superficial infections and healthy controls. Anti-staphylococcal IgG antibodies that reacted with two antigens of apparent molecular weight 110 and 98 kDa were specifically present in 96% (24/25) of patients with deep-seated infections, and were absent in, superficial and control sera. Moreover other Gram-positive and Gram-negative bacteria did not share these two unique antigens. The ELISA assays revealed significantly elevated levels of IgG antibodies to peptidoglycan (PG) in 18 of 25 (72%) patients with deep infection and 15 of 25 (60%) patients with superficial staphylococcal infection. The elevated levels of IgG antibodies to teichoic acid (TA) antigen were detected in all (100%) deep-seated group patients and among 40% (10/25) patients with superficial infection. An increase in levels of antibodies to PG showed a positive correlation trend with levels of IgG antibodies to TA only in deep infection group. Thus immunoblot detection of these two unique antigens as well as detection of elevated antibodies against PG and TA may be useful for the discrimination of staphylococcal deep-seated and superficial infection in humans.